Transcriptional glucocorticoid-response at the exon level and NR3C1 DNA-binding in childhood leukemia models: mRNA and ChIP-chip profiling
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE29063 | GEO | 2011/11/15
SECONDARY ACCESSION(S): PRJNA140245
REPOSITORIES: GEO
Similar Datasets
Project description:Glucorticoids (GCs) are steroid hormones with a wide range of physiological actions in different cell types and tissues. Their ability to induce apoptosis in malignant cells of the lymphoid lineage is exploited since decades in the treatment of childhood acute lymphoblastic leukemia (ALL), the molecular mechanism of this cell death induction being however still unknown. Using an integrative approach we delineated the transcriptional response of a preB- and a T-ALL model system employing Exon microarrays and identified in vivo interactions of the transcription factor GC receptor (GR) with genes' promoters in the same samples using the ChIP-on-chip technology. We did find differences in GR-DNA interaction in the genes' promoters between the two cell systems and also in extent and kinetics of GC mediated gene regulation. The overall response was stronger in CEM-C7H2 T-ALL cells where it was also accompanied by a stronger autoup-regulation of the GR gene. Considering the differing kinetics we furthermore defined a set of common response genes. These response genes were enriched in apoptosis related gene ontology processes, and about half of them yielded also GR enrichment in the respective promoter regions. Globally, the presence of GR at gene's promoters was higher for GC induced compared to repressed genes, indicating that GR mediates gene repression by interaction with distant enhancer regions or by cross-talk with other transcription factors. In order to identify the genes' GC regulated transcript variants we analyzed the data set also at the level of individual exons and predicted first exons of GC induced isoforms. We could thus define GC regulated variants of response genes, did however also identify first exons not corresponding to any known transcript. We reconfirmed these results for the genes ATP4B, GPR98, TBCD and ZBTB16 and identified for the latter also a novel exon employing 5' nested amplification of cDNA ends. Expression and regulation of this and all downstream exons of ZBTB16 was further verified by real time RT-PCR in 4 different preB-ALL cell lines. Thus, besides potential other novel gene isoforms, GCs induce a novel short transcript variant of ZBTB16 in preB-ALL cells. Finally, the implemented bioinformatic methods may be useful also for other high density microarray data sets to identify alternative splice events and transcript variant expression.
2011-11-15 | GSE29057 | GEO
Project description:Glucorticoids (GCs) are steroid hormones with a wide range of physiological actions in different cell types and tissues. Their ability to induce apoptosis in malignant cells of the lymphoid lineage is exploited since decades in the treatment of childhood acute lymphoblastic leukemia (ALL), the molecular mechanism of this cell death induction being however still unknown. Using an integrative approach we delineated the transcriptional response of a preB- and a T-ALL model system employing Exon microarrays and identified in vivo interactions of the transcription factor GC receptor (GR) with genes' promoters in the same samples using the ChIP-on-chip technology. We did find differences in GR-DNA interaction in the genes' promoters between the two cell systems and also in extent and kinetics of GC mediated gene regulation. The overall response was stronger in CEM-C7H2 T-ALL cells where it was also accompanied by a stronger autoup-regulation of the GR gene. Considering the differing kinetics we furthermore defined a set of common response genes. These response genes were enriched in apoptosis related gene ontology processes, and about half of them yielded also GR enrichment in the respective promoter regions. Globally, the presence of GR at gene's promoters was higher for GC induced compared to repressed genes, indicating that GR mediates gene repression by interaction with distant enhancer regions or by cross-talk with other transcription factors. In order to identify the genes' GC regulated transcript variants we analyzed the data set also at the level of individual exons and predicted first exons of GC induced isoforms. We could thus define GC regulated variants of response genes, did however also identify first exons not corresponding to any known transcript. We reconfirmed these results for the genes ATP4B, GPR98, TBCD and ZBTB16 and identified for the latter also a novel exon employing 5' nested amplification of cDNA ends. Expression and regulation of this and all downstream exons of ZBTB16 was further verified by real time RT-PCR in 4 different preB-ALL cell lines. Thus, besides potential other novel gene isoforms, GCs induce a novel short transcript variant of ZBTB16 in preB-ALL cells. Finally, the implemented bioinformatic methods may be useful also for other high density microarray data sets to identify alternative splice events and transcript variant expression.
2011-11-15 | GSE29056 | GEO
Project description:Glucorticoids (GCs) are steroid hormones with a wide range of physiological actions in different cell types and tissues. Their ability to induce apoptosis in malignant cells of the lymphoid lineage is exploited since decades in the treatment of childhood acute lymphoblastic leukemia (ALL), the molecular mechanism of this cell death induction being however still unknown. Using an integrative approach we delineated the transcriptional response of a preB- and a T-ALL model system employing Exon microarrays and identified in vivo interactions of the transcription factor GC receptor (GR) with genes' promoters in the same samples using the ChIP-on-chip technology. We did find differences in GR-DNA interaction in the genes' promoters between the two cell systems and also in extent and kinetics of GC mediated gene regulation. The overall response was stronger in CEM-C7H2 T-ALL cells where it was also accompanied by a stronger autoup-regulation of the GR gene. Considering the differing kinetics we furthermore defined a set of common response genes. These response genes were enriched in apoptosis related gene ontology processes, and about half of them yielded also GR enrichment in the respective promoter regions. Globally, the presence of GR at gene's promoters was higher for GC induced compared to repressed genes, indicating that GR mediates gene repression by interaction with distant enhancer regions or by cross-talk with other transcription factors. In order to identify the genes' GC regulated transcript variants we analyzed the data set also at the level of individual exons and predicted first exons of GC induced isoforms. We could thus define GC regulated variants of response genes, did however also identify first exons not corresponding to any known transcript. We reconfirmed these results for the genes ATP4B, GPR98, TBCD and ZBTB16 and identified for the latter also a novel exon employing 5' nested amplification of cDNA ends. Expression and regulation of this and all downstream exons of ZBTB16 was further verified by real time RT-PCR in 4 different preB-ALL cell lines. Thus, besides potential other novel gene isoforms, GCs induce a novel short transcript variant of ZBTB16 in preB-ALL cells. Finally, the implemented bioinformatic methods may be useful also for other high density microarray data sets to identify alternative splice events and transcript variant expression.
2011-11-15 | GSE29049 | GEO
Project description:Glucorticoids (GCs) are steroid hormones with a wide range of physiological actions in different cell types and tissues. Their ability to induce apoptosis in malignant cells of the lymphoid lineage is exploited since decades in the treatment of childhood acute lymphoblastic leukemia (ALL), the molecular mechanism of this cell death induction being however still unknown. Using an integrative approach we delineated the transcriptional response of a preB- and a T-ALL model system employing Exon microarrays and identified in vivo interactions of the transcription factor GC receptor (GR) with genes' promoters in the same samples using the ChIP-on-chip technology. We did find differences in GR-DNA interaction in the genes' promoters between the two cell systems and also in extent and kinetics of GC mediated gene regulation. The overall response was stronger in CEM-C7H2 T-ALL cells where it was also accompanied by a stronger autoup-regulation of the GR gene. Considering the differing kinetics we furthermore defined a set of common response genes. These response genes were enriched in apoptosis related gene ontology processes, and about half of them yielded also GR enrichment in the respective promoter regions. Globally, the presence of GR at gene's promoters was higher for GC induced compared to repressed genes, indicating that GR mediates gene repression by interaction with distant enhancer regions or by cross-talk with other transcription factors. In order to identify the genes' GC regulated transcript variants we analyzed the data set also at the level of individual exons and predicted first exons of GC induced isoforms. We could thus define GC regulated variants of response genes, did however also identify first exons not corresponding to any known transcript. We reconfirmed these results for the genes ATP4B, GPR98, TBCD and ZBTB16 and identified for the latter also a novel exon employing 5' nested amplification of cDNA ends. Expression and regulation of this and all downstream exons of ZBTB16 was further verified by real time RT-PCR in 4 different preB-ALL cell lines. Thus, besides potential other novel gene isoforms, GCs induce a novel short transcript variant of ZBTB16 in preB-ALL cells. Finally, the implemented bioinformatic methods may be useful also for other high density microarray data sets to identify alternative splice events and transcript variant expression.
2011-11-15 | GSE29003 | GEO
Project description:Glucocorticoids (GCs) are the key drugs for the treatment of pediatric acute lymphoblastic leukemia (ALL). Herein, investigation of the relationship between the N363S and BclI polymorphisms of the GC receptor gene (NR3C1) and the side effects of GCs during pediatric ALL therapy was aimed.N363S and BclI polymorphisms were analyzed in 49 patients with ALL treated between 2000 and 2012. The control group consisted of 46 patients with benign disorders. The side effects of GCs noted during the induction and reinduction periods were evaluated retrospectively according to the National Cancer Institute's Common Terminology Criteria for Adverse Events, version 4.0.The BclI allele and genotype frequencies were found similar in the two groups. No N363S polymorphism was detected in either of the groups. During induction, dyspepsia was found more frequently in the CG than in the CC (wild-type) genotype (36.4% vs. 5.3%, p=0.018) and depression symptoms more frequent in patients with the G allele (CG+GG) than the CC genotype (39.3% vs. 10.5%, p=0.031). During reinduction, Cushingoid changes, dyspepsia, and depression symptoms were more frequent in patients with the G allele (CG+GG) than in patients with the CC genotype (48.1% vs. 17.6%, p=0.041; 29.6% vs. 0.0%, p=0.016; 40.7% vs. 11.8%, p=0.040, respectively).In our study, patients with the BclI polymorphism were found to have developed more frequent side effects. We think that the BclI polymorphism should be considered while designing individualized therapies in childhood ALL.
| S-EPMC5440867 | biostudies-other
Project description:Patients undergoing major surgery experience postoperative inflammation, which may contribute to postoperative morbidity. Endogenous glucocorticoids (GCs) are an essential part of the stress response, but this response varies between individuals, which may in turn affect clinical outcome and specifically postoperative inflammation. Exon 1 of the NR3C1 gene, encoding the GC receptor (GR), contains an established region of differential regulation. DNA methylation patterns in this region have been found to differ between individuals. The present study investigated the methylation status and genotype in the cytosine‑phosphate‑guanine (CpG) island in exon 1 of NR3C1 in 24 patients [Median age 65.5 (range 42‑81) years, 11 male, 13 female] who underwent major abdominal (12 pancreatic, 12 hepatic) surgery and explored its association with postoperative complications. DNA was extracted from peripheral blood leukocytes and underwent targeted bisulfite sequencing of the CpG island. Complications were graded according to the Clavien‑Dindo classification and 14 out of 24 patients had postoperative complications. Multifactorial and partial least square analyses were used to analyse the data. A homogenous demethylated pattern was observed in all patients and no single CpG methylation was associated with postoperative complications. Four SNPs were significantly associated with higher Clavien‑Dindo scores. Genetic variability in the chromosome 5:143,402,505‑143,405,805 region of exon 1 of the GR gene NR3C1, but not DNA methylation, was associated with more severe postoperative complications in patients having major abdominal surgery. These results indicated that the patients' response to GCs may be of clinical importance for inflammatory conditions.
| S-EPMC9052000 | biostudies-literature
Project description:Glucocorticoids (GCs) are a central component in treating childhood acute lymphoblastic leukemia (chALL). They mainly act via regulating gene transcription. However, control of mRNA translation by GC has never been assessed systematically. In our research, T- and precursor B-ALL cells were cultured with and without GC for 6 hours and subjected to translational profiling, a technique combining sucrose gradient fractionation and microarray analysis of mRNA in different fractions. Analysis of GC regulation in different pools revealed no significant differences in regulation of mRNA translation by GC, suggesting no evidence for translational regulation by GC. Ribosome profiling by sucrose gradient fractionation followed by microarray analysis of RNA from 3 distinct pools representing free RNA (pool 1), RNA in small protein-RNA complexes or bound to single ribosomes (pool 2) and RNA from large protein-RNA complexes (polysomes, pool 3). See Material and Methods of the original article for a detailed description. Translational efficiency of genes was estimated by comparing the amount of a gene's mRNA in pool 3 with the total amount of mRNA of the gene (in all 3 pools). Translational regulation by GC was estimated by contrasting gene regulations in pool 3 with gene regulation between GC-treated and control samples in all 3 pools (depicted as transcriptional regulation). Moderated t-tests were used to assess significance for translational regulation.
2013-12-03 | E-GEOD-48680 | biostudies-arrayexpress
Project description:Glucocorticoids (GCs) are a central component in treating childhood acute lymphoblastic leukemia (chALL). They mainly act via regulating gene transcription. However, control of mRNA translation by GC has never been assessed systematically. In our research, T- and precursor B-ALL cells were cultured with and without GC for 6 hours and subjected to translational profiling, a technique combining sucrose gradient fractionation and microarray analysis of mRNA in different fractions. Analysis of GC regulation in different pools revealed no significant differences in regulation of mRNA translation by GC, suggesting no evidence for translational regulation by GC. Ribosome profiling by sucrose gradient fractionation followed by microarray analysis of RNA from 3 distinct pools representing free RNA (pool 1), RNA in small protein-RNA complexes or bound to single ribosomes (pool 2) and RNA from large protein-RNA complexes (polysomes, pool 3). See Material and Methods of the original article for a detailed description.
2013-12-03 | GSE48680 | GEO
Project description:Glucocorticoids (GCs) are natural stress induced steroid hormones causing cell cycle arrest and cell death in lymphoid tissues. Therefore they are the central component in the treatment of lymphoid malignancies, in particular childhood acute lymphoblastic leukemia (chALL). GCs act mainly via regulating gene transcription, which has been intensively studied by us and others. GC control of mRNA translation has also been reported but has never been assessed systematically. In this study we investigate the effect of GCs on mRNA translation on a genome-wide scale.Childhood T- (CCRF-CEM) and precursor B-ALL (NALM6) cells were exposed to GCs and subjected to "translational profiling", a technique combining sucrose-gradient fractionation followed by Affymetrix Exon microarray analysis of mRNA from different fractions, to assess the translational efficiency of the expressed genes. Analysis of GC regulation in ribosome-bound fractions versus transcriptional regulation revealed no significant differences, i.e., GC did not entail a significant shift between ribosomal bound and unbound mRNAs.In the present study we analyzed for the first time possible effects of GC on the translational efficiency of expressed genes in two chALL model systems employing whole genome polysome profiling. Our results did not reveal significant differences in translational efficiency of expressed genes thereby arguing against a potential widespread regulatory effect of GCs on translation at least in the investigated in vitro systems.
| S-EPMC4046653 | biostudies-literature
Project description:Treatment outcomes in children with acute lymphoblastic leukemia (ALL) have been improved substantially, with a cure rate exceeding 80% using conventional therapy. However, the outcome for patients with relapsed/refractory ALL remains unsatisfactory, despite the fact that these patients generally receive more intense therapy. Glucocorticoid (GC) resistance is a leading cause of treatment failure and relapse in ALL. Abnormal NR3C1 transcription and/or translation is strongly associated with GC resistance, but the underlying molecular mechanism and the clinical value of NR3C1 alterations with GC resistance in ALL treatment remain unclear. This study applied panel sequencing to 333 newly diagnosed and 18 relapsed ALL samples to characterize the link between NR3C1 and ALL further. We identified NR3C1 mutations in three patients with newly diagnosed ALL (0.9%) and two patients with relapsed ALL (11.1%). Functional analyses revealed that four of these five NR3C1 mutations (p. R477H, p. Y478C, p. P530fs, and p. H726P) were loss-of-function (LoF) mutations. A drug sensitivity test further showed that LoF NR3C1 mutations influence GC resistance. Saturated mutagenesis of hotspot R477 demonstrated the importance of this residue for NR3C1 function. The dominant-negative effect of p. R477C and p. R477S and the non-dominant negative effect of p. R477H and p. Y478C suggests multiple mechanisms underlying GC resistance. Thus, primary or acquired genomic lesions in NR3C1 may play a critical role in GC resistance and contribute to ALL treatment failure and/or relapse.
| S-EPMC8039513 | biostudies-literature